D-Lactic acid is a chiral, three-carbon organic acid, that bolsters the thermostability of polylactic acid. In this study, we
developed a microbial production platform for the high-titer production of D-lactic acid. We screened 600 isolates of lactic
acid bacteria (LAB) and identified twelve strains that exclusively produced D-lactic acid in high titers. Of these strains,
Lactobacillus saerimneri TBRC 5746 was selected for further development because of its homofermentative metabolism.
We investigated the effects of high temperature and the use of cheap, renewable carbon sources on lactic acid production and
observed a titer of 99.4 g/L and a yield of 0.90 g/g glucose (90% of the theoretical yield). However, we also observed L-lactic
acid production, which reduced the product’s optical purity. We then used CRISPR/dCas9-assisted transcriptional repression
to repress the two Lldh genes in the genome of L. saerimneri TBRC 5746, resulting in a 38% increase in D-lactic acid
production and an improvement in optical purity. This is the first demonstration of CRISPR/dCas9-assisted transcriptional
repression in this microbial host and represents progress toward efficient microbial production of D-lactic acid.
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L-alanine and L-anticapsin, is produced and excreted by
Bacillus subtilis under the control of quorum sensing. We
analyzed bacilysin-nonproducing strain OGU1 which was
obtained by bacA-targeted pMutin T3 insertion into the
parental strain genome resulting in a genomic organization
(bacA::lacZ::erm::bacABCDEF) to form an IPTG-inducible
bac operon. Although IPTG induction provided 3- to 5-fold
increment in the transcription of bac operon genes, no bacilysin
activity was detectable in bioassays and inability of the
OGU1 to form bacilysin was confirmed by UPLC-mass spectrometry
analysis. Phenotypic analyses revealed the deficiencies
in OGU1 with respect to colony pigmentation, spore coat
proteins, spore resistance and germination, which could be
rescued by external addition of bacilysin concentrate into its
cultures. 2DE MALDI-TOF/MS and nanoLC-MS/MS were
used as complementary approaches to compare cytosolic proteomes
of OGU1. 2-DE identified 159 differentially expressed
proteins corresponding to 121 distinct ORFs. In nanoLCMS/
MS, 76 proteins were differentially expressed in OGU1.
Quantitative transcript analyses of selected genes validated
the proteomic findings. Overall, the results pointed to the impact
of bacilysin on expression of certain proteins of sporulation
and morphogenesis; the members of mother cell compartment-
specific σE and σK regulons in particular, quorum
sensing and two component-global regulatory systems, peptide
transport, stress response as well as CodY- and ScoCregulated
proteins.
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