Youming Jiang,Xiaohan Ye,Tianwen Zheng,Weiliang Dong,Fengxue Xin,Jiangfeng Ma*,Min Jiang

State Key Laboratory of Materials-Oriented Chemical Engineering,College of Biotechnology and Pharmaceutical Engineering,Nanjing Tech University,Nanjing 211800,China

ABSTRACT L-malate is an intermediate of the tricarboxylic acid cycle which is naturally occurred in various microorganisms,and it has been widely applied in polymer,beverage and food,textile,agricultural and pharmaceutical industries.Driven by the pursuit of a sustainable economy,microbial production of L-malate has received much attention in last decades.In this review,we focus on the utilization of wastes and/or byproducts as feedstocks for the microbial production of L-malate.Firstly,we present the recent developments on the natural or engineered metabolic pathways that dedicate to the biosynthesis of L-malate,and also provide a comprehensive discussions on developing high-efficient producers.Then,the recent achievements in microbial production of L-malate from various carbon sources were concluded and discussed.Furthermore,some abundant non-food feedstocks which have been used for microbial production of other chemicals were reviewed,as they may be potential candidate feedstock for L-malate production in future.Finally,we outlined the major challenges and proposed further improvements for the production of L-malate.

Keywords:L-malate Renewable feedstock Microbial fermentation Bioeconomy C4 dicarboxylic acid

1.Introduction

L-malate is a C4 dicarboxylic acid and a crucial intermediate of tricarboxylic acid (TCA) cycle in the living cells.As one of important building-block and platform chemicals,it has been widely applied in polymer,beverage and food,textile,agricultural and pharmaceutical industries [1,2].In view of a wide range of commercial applications,L-malate has been identified as one of twelve most promising building-block chemicals that can be produced from biomass by the US Department of Energy [3].It had been reported that the annual world production of malate ranges from 60,000 to 200,000 tons,and a current global production capacity around 150,000 tons per year seems reasonable,estimated by one of the Chinese L-malate leading manufacturers Changmao Biochemical Engineering Company [4,5].With the ever-growing demand,it was expected that the global market of malate in 2024 will reach up to USD 240 million [6].

Traditionally,malate was produced by chemical methods through the hydration of maleic anhydride at a high temperature and pressure condition [7].However,a racemic mixture of the stereoisomers is generated as the form of D-(-)-and L-(+)-malate,which limits the application in the beverage and food as well as pharmaceutical industries due to that only L-(+)-isomer can be metabolized by living cells [1].Nowadays,biotechnology offers a reliable,feasible,environment friendly and sustainable route for converting renewable biomass to value-added chemicals,especially the chiral compounds.Thus,the shift towards a green economy of sustainable bio-based L-malate production from renewable substrate is getting more and more attention in last decades[7,8].However,it must be noted that the biotechnological process should be cost-competitive,which is the most crucial factor in the commercialization of bio-refinery process [9].

It had been reported that the material cost of fermentation accounts around 60% of total cost [10].Thus,utilization of waste biomass or low cost carbon sources is an efficient route to produce chemicals economically.In this review,we focus on the utilization of wastes and/or by-products as feedstocks for the microbial production of L-malate.Firstly,we present the recent developments on the natural or engineered metabolic pathways that dedicate to the biosynthesis of L-malate,and also provide a comprehensive discussions on developing high-efficient L-malate producers.Then,the recent achievements in microbial production of L-malate from various carbon sources were concluded and discussed.Furthermore,some potential non-food feedstocks which have been used for microbial production of other chemicals were reviewed,as they may be potential candidates feedstock for L-malate production.Finally,we outlined the major challenges and proposed further improvements for the production of L-malate based on metabolic engineering,synthetic biology and system metabolic engineering.

2.Metabolic Pathways for L-malate Production

Currently,rational design for engineering strains has brought a new broad perspective for microbial production of L-malate [11].The rapid developments of synthetic biology also provide a superior opportunity to accelerate the breeding of superior cell factories for production of L-malate with high yield,titer and productivity[12].

Up to now,four metabolic pathways for L-malate production from glucose have been reported,including (I) reductive TCA(rTCA) pathway;(II) oxidative TCA pathway;(III) one-step carboxylation with specific malic enzymes from pyruvate;(IV) noncyclic glyoxylate shunt route (Fig.1).

rTCA pathway is the major route for the biosynthesis of L-malate in the cytoplasm with a maximum theoretical yield of 2 mol·mol-1.It includes the carboxylation of pyruvate or phosphoenolpyruvate(PEP)to oxaloacetate (OAA)and further reduction to L-malate by malate dehydrogenase (MDH).Normally,this route occurs in various Fungi and yeast,such asAspergillus flavus[13],Penicillium sclerotiorum[14],Penicillium viticola[15].For example,asawell-knownproducer,A.flavusproduced 113 g·(L L-malate)-1with a high yield of 1.28 mol·mol-1by rTCA pathway [13].Also,a series of engineered microorganisms have been constructed by introduction and/or overexpression of enzymes in rTCA route for L-malate production.As an important model organism,Saccharomyces cerevisiaewas engineered for Lmalate production by co-overexpression of pyruvate carboxylase(PYC),cytosolic malate dehydrogenase (MDH) and malate permease (SpMAE) fromSchizosaccharomyces pombe,and 59.0 g·L-1of L-malate was produced with a yield of 0.31 g·g-1[16].Similar strategies were also adopted for engineeringAspergillus oryzae[17–19],Torulopsis glabrata[20]to enhance the biosynthesis of L-malate.Recently,an auxotrophicS.cerevisiaestrain thTAM was evaluated the efficiency of PYC fromA.flavus,MDH fromRhizopus oryzae,AfpycandRomdhwith higher specific activities,and modifiedSpmaeat different expression strengths.As a result,carbon flux distribution was optimized in strain W4209 with the Lmalate concentration,yield and productivity of 30.25 g·L-1,0.30 g·g-1and 0.32 g·L-1·h-1,respectively [21].

Fig.1.Four reported pathways for microbial production of L-malate from glucose.(I) Direct reduction of oxaloacetate via the reductive TCA pathway.(II) Formation from citrate via the oxidative TCA cycle.(III) Direct conversion of pyruvate via the one-step carboxylation.(IV) Formation from acetyl-CoA and oxaloacetate via the noncyclic glyoxylate route.OAA,oxaloacetate;MAL,L-malate;CIT,citrate;ICI,isocitrate;AKG,α-ketoglutarate;SUCC,succinyl-CoA;SUC,succinate;FUM,fumarate;PYR,Pyruvate;C1,;C2,acetyl-CoA;,maximum theoretical yield (in mol L-malate per mol glucose).

In addition to those eukaryotic microorganisms,Moonet al.introduced the PEP carboxykinase (encoded by thepckA) fromMannheimia succiniciproducensinto aptamutantE.coliWGS-10,and 9.25 g·(L L-malate)-1was obtained with a yield of 0.56 mol·mol-1[22].For further decreasing the by-products accumulation,genetic modification of intermediate competing pathways was carried out in succinate-producingE.colistrains by chromosomal inactivation.The recombinant accumulated 34.0 g·(L L-malate)-1with yield and productivity of 1.42 mol·mol-1and 0.47 g·L-1·h-1,respectively [23].As another important type strains,Bacillus subtilis168 was engineered by co-overexpression of PEP carboxylase fromE.coliand malate dehydrogenase fromS.cerevisiaeto construct a heterologous L-malate biosynthesis pathway.Combining with inactivation of lactate dehydrogenase,this engineered strain produced 15.65 mmol·L-1L-malate with a yield of 0.16 mol·mol-1[24].

The second route for production of L-malate is oxidative TCA pathway,which normally occurs in the mitochondrial or cytoplasmic TCA cycle under aerobic conditions[25].However,this oxidative pathway results in the release of CO2,limiting the maximum theoretical L-malate yield at 1 mol·(mol glucose)-1.Martinezet al.found anE.colimutant deficient in malate dehydrogenase produce 17.83 g·(L L-malate)-1in 47 h,with yield of 1.3 mol·mol-1and productivity of 0.38 g·L-1·h-1under aerobic conditionsviaoxidative TCA pathway [26].

One-step carboxylation with specific malic enzymes(ME) from pyruvate is the third pathway for the production of L-malate with maximum theoretical yield of 2 mol·mol-1[27].This pathway can directly convert pyruvate to L-malate by ME without intermediates generation,such as OAA.The capacity of ME from different species was evaluated in a pyruvate producing strainE.coliF0901,and the optimal NADP+-ME2fromArabidopsis thalianawas employed for the catalysis of one-step carboxylation from pyruvate to Lmalate.Then,pos5fromS.cerevisiaeencoding NADH kinase was further overexpressed to enhance the supply of NADPH by converting NADH to NADPH.Finally,21.65 g·(L L-malate)-1was produced with the yield of 0.36 g·g-1byE.coliF0931[27].However,16.54 g·(L pyruvate)-1was accumulated as the major byproduct,which might be due to that malic enzyme was more active for conversion of L-malate to pyruvate.To tackle this problem,a synthetic scaffold complex strategy has recently been employed by co-localizing pyruvate kinase (encoded bypykF) and NAD+-malic enzyme (encoded bysfcA) inE.colistrain to overcome the enzyme kinetics and push the reaction towards the production of L-malate [6].

The forth pathway for L-malate production is the noncyclic glyoxylate shunt route,in which OAA is replenished by PEP carboxylation with PPC or PCK,resulting in a maximum theoretical yield of 1.33 mol·mol-1.Gaoet al.systematically identified the ratelimiting step of noncyclic glyoxylate shunt route by in vitro modular optimization of multi-step cascade coordination.And rational pathway design was further adopted to shift the reaction equilibrium with controllable CRISPRi-guided multiplexed metabolic tuning [28].Finally,the engineered strainE.coliB0013-47 produced 36 g·(L L-malate)-1with a yield of 0.85 mol·mol-1.

3.Non-food feedstock and its application in L-malate production

Selection of suitable feedstock plays a significant role in the economics of biorefinery in addition to exploitation of the finest bio-catalysts.Up to now,most of the studies focused on using glucose as substrate for the production of bio-based products.However,glucose is typically obtained from starch which would not only take away the profit margins of biorefinery but also trigger the debate on food.Thus,numerous efforts have been paid on developing novel biorefinery processes based on non-food materials such as industrial wastes,lignocellulosic biomass and other inexpensive materials.The alternative raw materials compared with the glucose as substrate for microbial production of Lmalate are summarized in Table 1.

3.1.Crude glycerol from biodiesel industry

Glycerol is a promising feedstock for the microbial production of bio-based chemicals and fuels which is largely generated in the biodiesel industry [39].It is well known that the production of biodiesel has attracted attentions over the past decades which can serve as an environmentally alternative fuel for partial substitution of non-renewable petroleum-derived fuels[40].The market of biodiesel is estimated to reach 4.2 million tons in 2020 [41].In addition,10% waste glycerol (around 420,000 tons) generates in the process of biodiesel production,which greatly decreases the profit margin and economic feasibility[42].Fortunately,microbial conversions from crude glycerol to value-added products offers a brilliant solution to compensate for disposal-associated costs[39,40].

Generally,glycerol has a higher degree of reduction (k=4.67)which provides distinct advantage for biological conversion glycerol to fuels or more reduced chemicals at higher yield,compared to the use of normal carbon source such as glucose (k=4) [43].Microbial production of versatile chemicals had been intensively investigated using crude glycerol as sole carbon source in the past few years.For instance,it has been reported that the engineeredYarrowia lipolyticacould efficiently produce succinate from glycerol at low pH conditions [44].Anandet alevaluated the impact of crude glycerol on microbial growth and production of 1,3-propanediol byCitrobacter freundiiwith different grades of crude glycerol [41].A wild-typeClostridium pasteurianumGL11 has been identified,which produced 14.7 g·(L butanol)-1with a yield of 0.41 g·g-1from crude glycerol [45].E.colihas been rational engineered for effective conversion of crude glycerol to fuels and more reduced chemicals such as succinate[46],fumarate[47]and butanol [42].

Even though crude glycerol is an ideal feedstock,biodieselderived crude glycerol contains variable proportions of impurities depending on different biodiesel production process,such as methanol,ash,soap,salts,non-glycerol organic components and water,which might be toxic to the cells [48].Thus,mutagenesis and adaptive laboratory evolution (ALE) were adopted to enhance substrate uptake,and the strainUstilago trichophoraTZ1 was obtained which has significant potential for production of Lmalate from glycerol.With further optimization of medium and process parameters,the final titer,yield and overall production rate of L-malate in this mutant were up to incredible 118 g·L-1,0.26 g·g-1and 0.39 g·L-1·h-1,respectively [30].Moreover,metabolic engineering was established to further increase the efficiency of L-malate production by this organism.After overexpression of malate dehydrogenase(mdh2)with a shuttle plasmid,the recombinantU.trichophoraTZ1 Petef mdh2had an obvious lowered biomass formation,while the final titer,yield and specific production rate were enhanced to 134 g·L-1,0.36 g·g-1and 0.57 g·L-1·h-1[49].A.nigerstrains have been also used for L-malate production from glycerol fermentation [50].Iyyappanet al.investigated the effects of crude glycerol,spore inoculum and yeast extract concentrations,and shaking frequency on L-malate production ofA.nigerPJR1 and adopted the strategy of morphological control,leading to 83.2 g·(L L-malate)-1accumulation from 160 g·L-1crude glycerol within 192 h [32].

3.2.Thin stillage from bioethanol industry

In response to the rapidly growing demand for fuels,bioethanol has emerged as one of the most important liquid biofuels to replace gasoline [51].It was estimated that bioethanol production volume reached to 100 billion liters in 2015 [52].During the production of bioethanol,thin stillage is one of primary co-products[53].The majority of the remaining solids of thin stillage are glucan,xylan and glycerol[53].Thus,thin stillage can be used as ideal feedstock for microbial production of chemicals.Moreover,the remaining components such as crude protein,amino acids and minerals are also benefit for the cell growth [54].

Westet al.investigated the ability ofAspergillusstrains to utilize thin stillage for L-malate production.As a result,A.nigerATCC 9142 produced 17.0 g·(L L-malate)-1with a yield of 0.8 g·g-1from thin stillage[33,55].Considering the large volumes of thin stillage,the bioprocess of using the available sugar and glycerol in thin stil-lage for microbial production of L-malate has shown a great economic prospect and practical applicability.

Table 1 Overview of L-malate production by various microorganisms from different feedstocks.

3.3.Sugarcane/soybean molasses from food industry

Sugarcane is one of the largest global crop with approximately 1250 million tons per year,which is corresponding to almost 70%sugar supply worldwide [56,57].Sugarcane molasses is the main by-product containing approximately 50% mixed sugars (sucrose,glucose,and fructose) [58].

As wildE.colidoes not have the capability for sucrose metabolism,an invertase(CscA)fromE.coliW was successful displayed on the cell surface ofE.coliAFP111 by fusion with anchor protein OmpC to evaluate the metabolizing capabilities of sucrose and sugarcane molasses[59].After introduced cell surface display system,the engineered strain AFP111/pTrcC-cscAwas able to directly hydrolyze extracellular sucrose into glucose and fructose for cell growth and metabolism.Finally,41 g·L-1and 36.3 g·(L succinate)-1were produced with sucrose and sugarcane molasses,respectively.Fenget al.evaluated the L-malate production inAureobasidium pullulanswith sugarcane molasses as feedstock without any pretreatment or nutrient supplementation [60].After 140 h of fed-batch fermentation,94.2 g·L-1of L-malate was obtained by overexpressing PYC inA.pullulansstrain FJ-PYC with a yield of 0.62 g·g-1and a productivity of 0.67 g·L-1·h-1.S.cerevisiae,having natural ability for sucrose metabolism,can be employed as an ideal cell factory for the production of L-malate from sugarcane molasses based on previous engineering strategies [61].

Soybean is one of most important oilseed crop in the global trade of food,and the world annual production of soybeans is almost 320 million tons estimated by the US department of Agriculture(USDA)[62].Soy molasses is the major waste from soybean processing industry,and it contains significant amounts of sucrose and raffinose-family oligosaccharides (RFO) [63].Considering the high content of carbohydrate,various studies have been carried out for the production of chemicals.The feasibility of L-malate production from this feedstock was evaluated withA.pullulans,which is a yeast-like fungus having the ability to degrade plant materials and utilize various carbohydrates[34,63].The results showed that 71.9 g·(L L-malate)-1was obtained from soy molasses with an overall productivity above 0.29 g·h-1·L-1and a yield of 0.69 g·g-1inA.pullulansZX-10.

3.4.Lignocellulosic feedstocks

Lignocellulosic residues,which are estimated to exceed 2×1011tons per year worldwide are the largest waste source on the earth[64].However,most of them were discarded or burned.Since the utilization of such agricultural waste may greatly reduce the costs and provide a promising mode for the development of a sustainable and environment-friendly economy,a great deal of efforts and interests have been dedicated to this area [65].

The carbohydrate fraction of lignocellulose includes cellulose and hemicellulose,which could be further hydrolyzed to C6 (glucose,mannose,galactose) and C5 (xylose,arabinose) sugars by thermochemical or enzymatic hydrolysis as an ideal carbon source for the production of various chemicals and biofuels via microbial fermentation [66].For example,the second-generation bioethanol using lignocellulosic materials as biomass feedstock is receiving considerable attention as the great advantages of low cost,large availability and un-competitiveness with food resources [67].Recently,biosynthesis of L-malate from agricultural and forestry waste have been also intensively investigated [34,36,68].

Corn is the most widely cultivated crop,and corn stover is the major residues estimated almost 238 million tons per year [69].The dry matter of corn stover is mainly consisted of 20% cobs,10%husks,50%stalks and 20%leaves[70].As a low cost and abundant agricultural residue,corn stover predominantly contains cellulose (35%–45%),hemicellulose (20%–25%) and lignin (15%–20%)[71,72].

Zouet al.isolated the L-malate producer,A.pullulansCCTCC M2012223,by long-term adapted evolution in the aerobic fibrous bed bioreactors (AFBB).The adapted strain produced 38.6 g·(L Lmalate)-1with a productivity of 0.4 g·L-1·h-1from hydrolysate of corncob,and it could grow under the stress of 0.5 g·(L furfural)-1,3 g·(L hydroxymethylfurfural)-1,2 g·(L acetic acid)-1,and 0.5 g·(L formic acid)-1[73].Another strainA.pullulansYJ 6–11 was also evaluated with corncob hydrolysate,and 32.4 g·(L Lmalate)-1was obtained with a comparable or higher consumption rate of xylose than that of glucose [36].Moreover,Rhizopus DelemarHF-119 produced more than 120 g·(L L-malate)-1from 125 g·L-1biomass hydrolysate within 60 h,which is a striking progress for efficient production of L-malate from hydrolysate [37].Also,this strain has the ability of utilizing glucose and xylose simultaneously.Such characteristics provide this strain with superior performance for the utilization of various wasted biomass.

Even though significant progress has been made for biorefinery of lignocellulosic biomass,the high cost of hydrolysis especially the addition of expensive enzymes leads to high manufacturing cost.It is worth to note that some microorganisms are capable of synthesizing extracellular cellulase which can be applied to convert cellulose to L-malate directly.Deng and his coworkers introduced pyruvate carboxylase gene fromCorynebacterium glutamicuminto aThermobifida fuscamutant strain muC,and 62.76 g·(L Lmalate)-1was obtained by the recombinant strainT.fuscamuC-16 from cellulose directly with a yield of 0.63 g·g-1[35].

4.Other Potential Non-Food Feedstock for Microbial Production of L-malate

Here,a generally simplified representation of the process for biological production of L-malate from various wasted materials is shown in Fig.2.Some other renewable materials could be potential feedstocks for fermentation to various chemicals,including Lmalate production.

4.1.Whey from dairy industry

Fig.2.Diagrammatic illustration of the framework of biorefinery of L-malate.

Whey is an important renewable resource,which could be utilized as a low-cost substrate for the biotechnological production.Normally,whey is a major waste product of the dairy industry during yogurt,cheese,milk producing processes,and it mainly contains lactose (40–50 g·L-1) and casein (0.6%–0.8 %) [74].It has been reported that industrial cheese production occupies 26% of total European food waste or by-product streams [10].Such huge amounts of dairy industry waste combined with the advantages of low cost and especially non-competitiveness with food sources confer it to be an ideal feedstock for producing a series of valueadded chemicals and biofuels in a sustainable and cost-effective manner.Several effects have been successfully implemented in the production of bio-hydrogen[75],propionic acid[76]and lactulose [77]and other value-add products from whey.Even though there is no report on the production of L-malate from lactoseriched whey,the biorefinery process could be established by reprogramming some model strains using strategies such as metabolic engineering,synthetic biology.

4.2.Other lignocellulosic biomass

Generally,lignocellulosic biomass is primarily from agricultural and forestry residues(such as corn stover,wheat straw,rice straw)and processing byproducts (such as rice hush,sugarcane bagasse)[78,79].In addition to corn,rice and wheat are also important food crops in the world.After crops harvested,rice and wheat straws remain as major type of residues [80].It was reported that global production of rice and wheat straws are more than 730 and 354 million tons per year,respectively[51,81].Recently,the biological production of ethanol and other biofuels from crop straws has gained much attentions[82,83].As rice and wheat straw have similar percentages of components compared to corn stover,they could be potential feedstocks for L-malate production.

Forestry processing industry is another major resource that generates vast amounts of lignocellulosic biomass.They are normally treated as fuels to produce bioenergy in the form of heat and electricity[84].Forestry residues are composed mainly of cellulose(33%–51%),hemicelluloses(20%–30%)and lignin(21%–32%),and the composition of each residue is varying from the tree species [85].So far,most of researches focused on the production of ethanol via fermentation by using various microorganisms suchS.cerevisiaeandE.coli.Recently,the feasibility of producing fumarate fromEucalyptus globuluswood hydrolysates byRhizopus arrhizusDSM5772 was confirmed [86].Under optimized conditions,highest fumarate titer (9.84 g·L-1) and yield (0.44 g·g-1) were obtained from the mixtures of ion-exchange treated hydrolysates.Kimet al.also investigated the performance ofMannheimia succiniciproducensMBEL55E for the production of succinate from wood hydrolysates with batch and continuous fermentation [87].Finally,the titer,yield and productivity of succinate in batch and continuous cultures reached to 11.73 and 7.98 g·L-1,0.56 and 0.55 g·(g total sugar)-1(glucose and xylose),as well as 1.17 and 3.19 g·L-1·h-1,respectively.Until now,there are no reports related to the production of L-malate from forestry residues.Consequently,more attempts can be tried on biological production of L-malate from wasted forestry residues based on the success of fumarate and succinate production.

In the sugarcane industry,135 kg of sugar and 130 kg of bagasse were produced from each ton of sugarcane,and almost 220 million tons of bagasse were generated per year [88].The usage of sugarcane bagasse is typically limited as a fuel for thermal energy generation and for cogeneration of electricity [89].However,the high cellulose (40%–45%) and hemicellulose (30%–35%) content of sugarcane bagasse can be readily hydrolyzed into fermentable sugars and then converted to a series of chemicals [90].In the past few years,systematic studies had been carried out for large-scale biological production of ethanol from sugarcane bagasse by reduction of hydrolysis costs,optimization of process,and development of host microorganism with stable performance [90].In addition to biofuels,biological production of fine chemicals from sugarcane bagasse is another research direction.For example,18.88 g·(L succinate)-1was produced from sugarcane bagasse hydrolysate with a high yield of 0.89 g·(L total sugar)-1by a metabolic engineeredE.colistrain BA204,demonstrating the great potential of using sugarcane bagasse as feedstock [91].

5.Conclusions

In this study,four metabolic pathways for the production of Lmalate were discussed.Among these routes,rTCA pathway appears to be the most efficient route for the microbial production of Lmalate,attributing to the less reactions and high activities.For further decrease the cost of biological processes,a large variety of carbon sources which had been reported for the bio-based production of L-malate were summarized.Moreover,this review also assessed the future potential of utilizing these renewable wastes for biological production of L-malate.

Even a great deal of progress had been made in the microbial engineering and process engineering as well as the exploration of low cost alternative feedstocks,it should be mentioned that there are still many hurdles having not been overcame in the commercialization of microbial L-malate production.For example,the high cost of enzymes for hydrolysis during feedstock pretreatment process limits the commercial production of L-malate.An alternative solution is developing novel cellulose-degrading enzymes with higher activity or adopting an integrated biorefinery strategy in which hydrolyzes of cellulose is consolidated with the direct utilization of the produced sugars by a single microorganism with cellulolytic enzymes producing ability.Another critical issue is that most of the native L-malate producers are suffered from low tolerance to substrates(i.e.dilute-acid lignocellulosic hydrolysates)and high level of L-malate.Here,we suggest to adopt the strategies of mutagenesis,ALE and rational engineering to improve cell tolerance to toxic components,which can partially relieve the inhibitory effect and increase the titer and productivity.Also,product recovery and downstream processing occupy a large fraction of production costs in biorefinery.

Nevertheless,utilization of waste materials as substrate for production of L-malate has considerable potential.With the development of excellent microbial cell factories and exploitation of lowcost feedstocks,a more economical and sustainable process can be achieved by metabolic engineering strategies and bioprocess techniques from the perspective of “green chemistry”and “sustainable economy”.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

This work was supported by the National Key R&D Program of China(2018YFA0901500),the National Natural Science Foundation of China (21706124,21727818),the Key Science and Technology Project of Jiangsu Province (BE2016389),and the Jiangsu Synergetic Innovation Center for Advanced Bio-Manufacture of China.