High Yields of 2,3-Butanediol and Mannitol in Lactococcus lactis through Engineering of NAD ⁺ Cofactor Recycling

Abstract

Manipulation of NADH-dependent steps, and particularly disruption of the las-located lactate dehydrogenase (ldh) gene in Lactococcus lactis, is common to engineering strategies envisaging the accumulation of reduced end products other than lactate. Reverse transcription-PCR experiments revealed that three out of the four genes assigned to lactate dehydrogenase in the genome of L. lactis, i.e., the ldh, ldhB, and ldhX genes, were expressed in the parental strain MG1363. Given that genetic redundancy is often a major cause of metabolic instability in engineered strains, we set out to develop a genetically stable lactococcal host tuned for the production of reduced compounds. Therefore, the ldhB and ldhX genes were sequentially deleted in L. lactis FI10089, a strain with a deletion of the ldh gene. The single, double, and triple mutants, FI10089, FI10089⌬ldhB, and FI10089⌬ldhB⌬ldhX, showed similar growth profiles and displayed mixed-acid fermentation, ethanol being the main reduced end product. Hence, the alcohol dehydrogenase-encoding gene, the adhE gene, was inactivated in FI10089, but the resulting strain reverted to homolactic fermentation due to induction of the ldhB gene. The three lactate dehydrogenase-deficient mutants were selected as a background for the production of mannitol and 2,3-butanediol. Pathways for the biosynthesis of these compounds were overexpressed under the control of a nisin promoter, and the constructs were analyzed with respect to growth parameters and product yields under anaerobiosis. Glucose was efficiently channeled to mannitol (maximal yield, 42%) or to 2,3-butanediol (maximal yield, 67%). The theoretical yield for 2,3-butanediol was achieved. We show that FI10089⌬ldhB is a valuable basis for engineering strategies aiming at the production of reduced compounds.Lactococcus lactis, a fermentative bacterium used worldwide in the manufacture of dairy products, is among the best characterized species of lactic acid bacteria (LAB). The wealth of knowledge generated in the fields of lactococcal genetics and physiology, combined with a "generally recognized as safe" (GRAS) status, a relatively simple metabolism, and a small genome, has rendered L. lactis an attractive model with which to implement metabolic engineering strategies (12,21,53).L. lactis is a homofermentative bacterium, which converts approximately 95% of the sugar substrate to lactic acid. In the last 15 years, numerous attempts have been made to reroute carbon flux from lactate to the production of other organic compounds via metabolic engineering. Manipulation of NADH-dependent steps is common to many of the strategies envisaging such a goal. In particular, disruption of the las (lactic acid synthesis) operonencoded lactate dehydrogenase (LDH), the major player in the regeneration of NAD ϩ

Synthesis of (3R)-acetoin and 2,3-butanediol isomers by metabolically engineered Lactococcus lactis

“…The maximum titers of (3 R )-acetoin, R-BDO and m-BDO obtained in this study were 27, 43 and 51 g/L respectively and the yield achieved was around 78 to 89% of theoretical maximum. The yield of 2,3-BDO achieved in this study for L. lactis was higher than obtained by Gaspar et al 22. (67% of theoretical maximum), where formate and ethanol were additional by-products.…”

“…It has previously been shown that when butA was over-expressed in an engineered L. lactis strain, m-BDO could be formed22. To completely eliminate m-BDO formation and achieve (3 R )-acetoin as the sole fermentation product, we therefore further inactivated the native butB and butA genes, which are co-located in an operon, thus generating VJ017 (MG1363 Δ 3 ldh Δ pta Δ adhE Δ butBA ) (Table 3).…”

Blue-light-dependent inhibition of twitching motility in Acinetobacter baylyi ADP1: additive involvement of three BLUF-domain-containing proteins

“…Exceptions are e.g. the seven rrn operons in E. coli (Stevenson & Schmidt, 2004), the four lactate dehydrogenases in Lactococcus lactis (Gaspar et al, 2011), and the three tcpD detoxification genes for the degradation of haloaromatic compounds in Cupriavidus necator (Pérez-Pantoja et al, 2009). In most cases there is a clear physiological function for this redundancy: high transcript levels that cannot be provided with a single promoter (in E. coli and Lactococcus), and differentiated selectivity of the separate promoters, or the advantage of rapid response in environments defined by fluctuations in resource availability, in the case of Cupriavidus.…”

Alcohol dehydrogenase activity in Lactococcus chungangensis: Application in cream cheese to moderate alcohol uptake

“…Many studies have detailed the ADH gene and its activity for Lactococcus lactis ssp. lactis in the field of sugar and ethanol production (Even et al, 2001;Palmfeldt et al, 2004;Gaspar et al, 2011;Solem et al, 2013;Costa et al, 2014;Gänzle, 2015).…”